Ascertaining the structure and function of various biochemical and nonbiochemical molecules and materials is becoming increasingly important to chemists and biochemists alike. With the development of new areas of research, novel, efficient and effective analysis and measurement tools are in high demand. For instance, with increasing developments in proteomics, new techniques are being developed and designed each day for sequencing and ascertaining primary structure of proteins and peptides. Although there are a number of techniques to do this, none are as precise, efficient and effective as mass spectrometry.
Ascertaining the amino acid sequence of a peptide is an important task because the amino acid sequence of the peptide or protein often determines higher order structure as well as function. In addition, the peptide structure can be used to search protein databases to determine where the peptide originated from. Since peptides can be quite large, often times these molecules are fragmented by mass spectrometry techniques and the resulting abundance and sequence data can later be determined or de-convoluted with the help of libraries. It is ideal for fragmentation to occur at all the amide bonds in the peptide backbone such that a /b-series or y-series is generated. This greatly aids sequence interpretation. Presently, there are a number of techniques for performing fragmentation of molecules in mass spectrometry systems. Each of these methods suffers from some limitations.
Collision induced dissociation (CID) is a technique in which a peptide or other molecule is accelerated into a gas and is fragmented by collisions with the gas molecules. CID is limited in the sense that it does not allow specificity in the fragmentation of certain bonds. There are no electronic transitions in the molecules to be fragmented and only gas may be employed with the collisions. The technique is generally effective for fragmenting different molecules, but provides limited specificity in the case of peptides.
Infrared photo-dissociation (IRPD) is another fragmentation technique which employs infrared photons to excite the translational, rotational and vibrational bonds of molecules. The technique is effective in providing information regarding various molecules, structures and bonds. However, since there are no electronic transitions, the technique is generally ineffective in fragmenting peptides or other molecules along specific bonds. No gas is used with this technique. Long wavelengths are employed to fragment molecules.
Surface induced dissociation (SID) is a technique that accelerates the molecule or peptide into a surface. The technique does not require a gas and the fragmentation of bonds is non-specific.
Electron capture dissociation (ECD) is another technique known and used in the art. This technique works by capturing an electron on the surface of charged molecules. In other words, EDC occurs wherein dissociation of the analyte molecules and ions results from attachment of low energy electrons. This technique provides non-specific bond fragmentation. The technique is limited, however, in that it only works on multiple charged ions and only in the positive mode.
Electron transfer dissociation (ETD) is the newest technique now being heavily used to study molecules. In this technique, a thermal electron is transferred from a negative ion to the analyte ion as a means of transferring energy to destabilize the ions. This technique was designed and developed particularly for peptide fragmentation. It is effective in providing simple fragmentation patterns that are easily understandable. The technique is limited, however, in that it only works on multiple charged ions and only in the positive mode.
In summary, there is a need for an apparatus and method to efficiently, effectively and accurately fragment different molecules for mass spectrometry analysis. In addition, there is a need to be able to fragment peptides at bond specific points for quick, efficient and easy determination of amino acid sequences. These and other problems are addressed by the present invention.